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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2018 Annual Report of the Division of Intramural Research

Regulation of Mammalian Cell Proliferation and Differentiation

Melvin DePamphilis
  • Melvin L. DePamphilis, PhD, Head, Section on Eukaryotic Gene Regulation
  • Alex Vassilev, PhD, Staff Scientist
  • Xiaohong Zhang, BA, Technical Assistant
  • Gaurav Sharma, PhD, Visiting Fellow
  • Arup Chakraborty, PhD, Research Fellow
  • Sushil Jaiswal, PhD, Postdoctoral Fellow
  • Ajit Roy, PhD, Postdoctoral Fellow
  • Constandina O'Connell, BS, Postbaccalaureate Fellow
  • John Oh, BS, Postbaccalaureate Fellow

Nothing is more fundamental to living organisms than the ability to reproduce. Each time a human cell divides, it must duplicate its genome, a problem of biblical proportions. A single fertilized human egg contains 2.1 meters of DNA. An adult of about 75 kg (165 lb) consists of about 29 trillion cells containing a total of about 60 trillion meters of DNA, a distance equal to 400 times that of Earth to sun. Not only must the genome be duplicated trillions of times during human development, but it must be duplicated once and only once each time a cell divides (termed mitotic cell cycles). If we interfere with this process by artificially inducing cells to re-replicate their nuclear genome before cell division, the result is DNA damage, mitotic catastrophe, and programmed cell death (apoptosis). On rare occasions, specialized cells can duplicate their genome several times without undergoing cell division (termed endocycles), but when this occurs, it generally results in terminally differentiated polyploid cells, which are viable but no longer proliferate. However, as we age, the ability to regulate genome duplication diminishes, resulting in genome instability, which allows genetic alterations that can result in promiscuous cell division—better known as cancer.

Our research program focuses on three questions: the nature of the mechanisms that restrict genome duplication to once per cell division; how these mechanisms are circumvented to allow developmentally programmed induction of polyploidy in terminally differentiated cells; and how we can manipulate these mechanisms to destroy cancer cells selectively.

Links between DNA replication, stem cells, and cancer

Cancers can be categorized into two groups: those whose frequency increases with age and those resulting from errors during mammalian development. The first group is linked to DNA replication through the accumulation of genetic mutations that occur during proliferation of developmentally acquired stem cells that give rise to and maintain tissues and organs. The mutations, which result from DNA replication errors as well as environmental insults, fall into two categories: cancer-driver mutations that initiate carcinogenesis, and genome-destabilizing mutations that promote aneuploidy through excess genome duplication and chromatid mis-segregation. Increased genome instability results in accelerated clonal evolution, leading to the appearance of more aggressive clones with increased drug resistance. The second group of cancers, termed germ-cell neoplasia, result from the mislocation of pluripotent stem cells during early development. During normal development, pluripotent stem cells that originate in early embryos give rise to all cell lineages in the embryo and adult, but when they mislocate to ectopic sites they produce tumors. Remarkably, pluripotent stem cells, like many cancer cells, depend on the geminin protein to prevent excess DNA replication from triggering DNA damage–dependent apoptosis. The link between the control of DNA replication during early development and germ cell neoplasia reveals geminin as a potential chemotherapeutic target in the eradication of cancer progenitor cells.

Geminin is essential for pluripotent cell viability during teratoma formation, but not for differentiated cell viability during teratoma expansion.

Pluripotent embryonic stem cells (ESCs) are unusual in that geminin has been reported to be essential either to prevent differentiation by maintaining expression of pluripotency genes or to prevent DNA re-replication–dependent apoptosis. To distinguish between these two incompatible hypotheses, we inoculated immune-compromised mice subcutaneously with ESCs harboring conditional Gmnn alleles alone or together with a tamoxifen-dependent Cre recombinase gene. We then injected the mice with tamoxifen at various times, during which the ESCs proliferated and differentiated into a teratoma. For comparison, the same ESCs were cultured in vitro in the presence of monohydroxytamoxifen. The results revealed that geminin is encoded by a haplosufficient gene that is essential for ESC viability before the cells differentiate into a teratoma, but once a teratoma is established, the differentiated cells can continue to proliferate in the absence of Gmnn alleles, geminin protein, or pluripotent stem cells. Thus, differentiated cells did not require geminin for efficient proliferation within the context of a solid tissue, although they did when teratoma cells were cultured in vitro. The results provide proof-of-principle that preventing geminin function could prevent malignancy in tumors derived from pluripotent cells by selectively eliminating the progenitor cells with little harm to normal cells.

DHS (trans-4,4′-dihydroxystilbene) suppresses DNA replication and tumor growth by inhibiting ribonucleotide reductase regulatory subunit M2 (RRM2).

Given that inhibition of DNA replication can lead to replication fork stalling, resulting in DNA damage and apoptotic death, inhibitors of DNA replication are commonly used in cancer chemotherapy. Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the biosynthesis of deoxyribonucleoside triphosphates (dNTPs), which are essential for DNA replication and DNA–damage repair. Gemcitabine, a nucleotide analog that inhibits RNR, has been used to treat various cancers. However, patients often develop resistance to this drug during treatment. Thus, the development of new drugs that inhibit RNR is needed. We identified a synthetic analog of resveratrol (3,5,4′-trihydroxy-trans-stilbene), termed DHS (trans-4,4'-dihydroxystilbene), that acts as a potent inhibitor of DNA replication. Molecular docking analysis identified the RRM2 of RNR as a direct target of DHS. At the molecular level, DHS induced cyclin F–mediated down-regulation of RRM2 by the proteasome. Thus, treatment of cells with DHS reduced RNR activity and consequently the synthesis of dNTPs, with concomitant inhibition of DNA replication, arrest of cells at S-phase, DNA damage, and finally apoptosis. In mouse models of tumor xenografts, DHS was efficacious against pancreatic, ovarian, and colorectal cancer cells. Moreover, DHS overcame both gemcitabine resistance in pancreatic cancer and cisplatin resistance in ovarian cancer. Thus, DHS is a novel anti-cancer agent that targets RRM2 with therapeutic potential either alone or in combination with other agents to arrest cancer development.

CDK1 inhibition facilitates formation of syncytiotrophoblasts and expression of human chorionic gonadotropin.

Human placental syncytiotrophoblast (STB) cells play essential roles in embryo implantation and nutrient exchange between the mother and the fetus. STBs are polyploid cells, which are formed by fusion of diploid cytotrophoblast (CTB) cells. Abnormality in STB formation can result in pregnancy-related disorders. While several genes have been associated with CTB fusion, the initial events that trigger cell fusion are not well understood. Our primary objective was to enhance our understanding of the molecular mechanism of placental cell fusion.

We used FACS (fluorescence-activated cell sorting) and microscopic analysis to optimize Forskolin-induced fusion of BeWo cells (surrogate of CTBs) and we subsequently analyzed changes in the expression of various cell-cycle regulator genes through Western blotting and qPCR. We performed immunohistochemistry on first-trimester placental tissue sections to validate the results in the context of placental tissue. We studied the effect of the cyclin-dependent kinase 1 (CDK1) inhibitor RO3306 on BeWo cell fusion using microscopy and FACS, and we monitored the expression of human chorionic gonadotropin (hCG) with Western blotting and qPCR (quantitative polymerase chain reaction).

The data showed that the placental cell fusion was associated with down-regulation of CDK1 and its associated cyclin B, and significant reductions in DNA replication. Moreover, inhibition of CDK1 by an exogenous inhibitor induced placental cell fusion and expression of hCG. We thus showed that placental cell fusion can be induced by inhibiting CDK1. The study has a high therapeutic significance to manage pregnancy related abnormalities.

A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt several events in lysosome homeostasis.

High-throughput screening identified five chemical analogs (termed the WX8-family) that disrupted three events in lysosome homeostasis: (1) lysosome fission via tubulation without preventing homotypic lysosome fusion; (2) trafficking of molecules into lysosomes without altering lysosomal acidity; and (3) heterotypic fusion between lysosomes and autophagosomes. Remarkably, these compounds did not prevent homotypic fusion between lysosomes, despite the fact that homotypic fusion required some of the same machinery essential for heterotypic fusion. The effects varied 400-fold among WX8–family members, were time- and concentration-dependent, reversible, and resulted primarily from their ability to bind specifically to the PIKFYVE phosphoinositidekinase. The ability of the WX8 family to prevent lysosomes from participating in autophagy suggested that they have therapeutic potential in treating autophagy-dependent diseases. In fact, the most potent family member (WX8) was 100-times more lethal to ‘autophagy-addicted’ melanoma A375 cells than the lysosomal inhibitors hydroxychloroquine and chloroquine. In contrast, cells that were insensitive to hydroxychloroquine and chloroquine were also insensitive to WX8. Therefore, the WX8 family of PIKFYVE inhibitors provides a basis for developing drugs that could selectively kill autophagy-dependent cancer cells, as well as increasing the effectiveness of established anti-cancer therapies through combinatorial treatments.

Publications

  1. Sharma G, Guardia CM, Roy A, Alex Vassilev A, Saric A, Griner LN, Marugan J, Ferrer M, Bonifacino JS, DePamphilis ML. A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis. Autophagy 2018;in press.
  2. Chen CW, Li Y, Hu S, Zhou W, Meng Y, Li Z, Zhang Y, Sun J, Bo Z, DePamphilis ML, Yen Y, Han Z, Zhu W. DHS (trans-4,4'-dihydroxystilbene) suppresses DNA replication and tumor growth by inhibiting RRM2 (ribonucleotide reductase regulatory subunit M2). Oncogene 2018;Epub ahead of print.
  3. Ullah R, Dar S, Ahmad T, de Renty C, Usman M, DePamphilis ML, Faisal A, Shahzad-Ul-Hussan S, Ullah Z. CDK1 inhibition facilitates formation of syncytiotrophoblasts and expression of human chorionic gonadotropin. Placenta 2018;66:57-64.
  4. Vassilev A, DePamphilis ML. Links between DNA replication, stem cells and cancer. Genes (Basel) 2017;8(2):E45.
  5. Adler-Wailes DC, Kramer JA, DePamphilis ML. Geminin is essential for pluripotent cell viability during teratoma formation, but not for differentiated cell viability during teratoma expansion. Stem Cells Dev 2017;26(4):285-302.

Collaborators

  • Juan Bonifacino, PhD, Section on Intracellular Protein Trafficking, Bethesda, MD
  • Marc Ferrer, PhD, Chemical Genomics Center, NCATS, Bethesda, MD
  • Juan Marugan, PhD, Division of Pre-Clinical Innovation, NCATS, Bethesda, MD
  • Zakir Ullah, PhD, Lahore University of Management Sciences, Lahore, Pakistan
  • Wenge Zhu, PhD, George Washington University Medical School, Washington, DC

Contact

For more information, email depamphm@mail.nih.gov or visit http://depamphilislab.nichd.nih.gov.

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